SUMMARY/ABSTRACT Reverse genetic techniques have revolutionized biology over the past 30 years, a revolution that has mostly passed the study of the human filarial parasites by. This is a result of the difficult biology of these organisms. They do not survive outside of their vertebrate hosts, and mechanical transfection methods result in damage to the parasite cuticle, which in turn results in the death of the parasite when it is re-introduced into its host. Despite these limitations, some progress in employing reverse genetics to these parasites has been made. Roughly 15 years ago, our laboratory developed a method for biolistic transfection of isolated B. malayi embryos. Although the embryos are not developmentally competent, we used this technique to identify both core promoter domains and regulatory regions in B. malayi promoters. While these studies were useful in defining some general aspects of promoter structure, transcriptional regulation and RNA processing in B. malayi, the technology cannot be applied to the most medically important and biologically interesting life cycle stages of the parasite. To apply reverse genetics to these stages requires a method to integrate exogenous sequences into the B. malayi genome, so that stably transfected parasite lines can be produced. We have recently taken advantage of a novel culture method that allows us to efficiently produce transgenic developmentally competent L4 larvae of B. malayi. We have built upon this advance and the knowledge we have gained from 15 years of work with our transient transfection system to develop a piggyBac transposon based transfection method that results in the stable integration of transgenes into the B. malayi genome. While this represents a significant advance, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which permits precise targeting (and editing) of particular sequences in the genome. The overall goal of this proposal will be to exploit our advances in stable transfection of B. malayi to develop a CRISPR-CAS9 based toolkit capable of routine and efficient production of stably transfected parasite lines. To accomplish this overall goal, the following aims are proposed: 1. To develop a CRISPR-CAS9 gene-editing system for B. malayi. 2. To validate this technology with a preliminary study of the spatial expression of two genes in B. malayi. 3. To develop methods for genotyping, isolation of isogenic lines and long-term preservation of transgenic parasites.